Apparatus for immune analysis

ABSTRACT

A total reflection cell has a total reflection prism on at least one surface thereof. A mixed solution containing a gold colloid labelled antibody which is adsorbed on a gold colloid is stored in the total reflection cell, and a sample solution containing an antigen causing antigen-antibody reaction with the antibody is added thereto for forming a gold colloid labelled immune complex. A measuring beam is introduced into the total reflection prism from an incident optical system at an angle θ of incidence causing total reflection and an outgoing beam from the total reflection prism is received by a measuring optical system, thereby measuring absorption by the gold colloid labelled immune complex and carrying out qualification and determination of the antigen.

This application is a continuation-in-part of application Ser. No. 08/592,522, filed Jan. 26, 1996, now U.S. Pat. No. 5,750,410, the benefit of which is hereby claim under 35 U.S.C. § 120.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of and apparatus for immunologically analyzing an object of measurement in the field of clinical testing, biochemical sample measurement, quality control of drugs and the like.

2. Description of the Background Art

Immunological analysis methods employing antigen-antibody reaction include fluorescent immunoassay and luminous immunoassay. In any one of these methods, an antigen which is labeled with a fluorescent material or a chemiluminescent material is reacted to an antibody with an antigen which is an object of measurement in competition, to form an immune complex which is a molecular complex by the antigen-antibody reaction for measurement fluorescence or luminescence from the label, thereby quantitatively analyzing the target material.

On the other hand, a method of adding an antigen of an antibody of an object of measurement to an antibody or an antigen and measuring absorption or scattering of light by an immune complex which is formed by antigen-antibody reaction thereby making quantitative analysis is known as an optical measuring method utilizing neither fluorescence nor luminescence. Quantitative analysis methods utilizing light scattering phenomenona include turbidimetry and nephelometry. The former is adapted to measure transmitted light which is attenuated by absorption and scattering, while the latter is adapted to measure scattering light intensity. The method by light scattering is adapted to measure Rayleigh scattering or Mie scattering, in response to sizes of particles as measured.

A fluorescent immunoassay apparatus belonging to fluorescent immunoassay is proposed as an immunoassay apparatus employing a total reflection cell (refer to Japanese Patent Laying-Open Gazette No. 5-203574 (1993)). In this fluorescent immunoassay, an antibody is fixed to a surface of a total reflection prism, an antigen contained in a sample is bonded to the antibody by antigen-antibody reaction, and an antibody which is labelled with a fluorescent material is further bonded to the antigen by antigen-antibody reaction. Thereafter B-F separation is performed to remove an unreacted antibody which is labelled with the fluorescent material, and then an excitation beam is introduced into the total reflection prism to excite the labelled antigen-antibody immune complex which is constrained on the surface of the total reflection prism, for measuring fluorescence generated from the fluorescent material.

The method utilizing light scattering, which is homogeneous immunoassay, is a simple method requiring neither B-F separation nor washing. However, the method utilizing Rayleigh scattering or Mie scattering has problems of low detection sensitivity and low measuring accuracy in relation to a low concentration material.

On the other hand, the fluorescent or luminescent immunoassay requires complicated chemical treatment for labelling an antigen or an antibody with a fluorescent or chemiluminescent material. Further, most thereof is heterogeneous immunoassay, which inevitably requires B-F separation for separating an immune complex (B) making antigen-antibody reaction from an antigen or antibody (F) making no antigen-antibody reaction and washing through a large number of analytical steps.

SUMMARY OF THE INVENTION

The inventors have made deep study in order to qualitatively and quantitatively analyze an immune complex by absorption measurement of the immune complex, to discover that absorption of the immune complex can be measured in excellent sensitivity when the immune complex is brought into a state adsorbed by noble metal colloidal particles, although no absorption having sensitivity suitable for measurement can be obtained from the immune complex itself.

An object of the present invention is to make it possible to qualify or further determine an immune material by absorption measurement of an immune complex further easily as compared with fluorescent or luminescent immunoassay.

An immune analysis method according to the present invention includes the steps of mixing a solution containing an antibody or an antigen which is labelled with noble metal colloidal particles with a sample solution to form a sample mixed solution for reacting an antigen or an antibody contained in the sample solution with the labelled antibody or antigen thereby forming a noble metal colloidal labelled immune complex, and employing a cell having a total reflection prism at least on one surface thereof and introducing a measuring beam of a wavelength in a range from visible to infrared regions into the total reflection prism at an angle of incidence causing total reflection thereby measuring adsorption of the measuring beam caused at the interface between the total reflection prism and the sample mixed solution. Thus, the antigen or the antibody which is contained in the sample is qualified or further determined.

The noble metal colloid can be prepared from a colloid of gold, silver or copper, and a proper grain size thereof is 5 to 50 nm.

According to the present invention, absorption measurement by the measuring beam can be carried out without separating the antibody or the antigen which is labelled with the noble metal colloid or an unreacted one of the antigen or the antibody contained in the sample solution, thereby implementing homogenous immunoassay analysis. Besides, high sensitive measurement can be implemented by using the noble metal colloid.

An immunoassay apparatus according to the present invention, which is adapted to implement the aforementioned method, comprises a total reflection cell having a total reflection prism consisting of a material having a larger refractive index than the sample mixed solution on at least one of wall surfaces defining a space in which the sample mixed solution is present, an incident optical system for introducing a measuring beam of a wavelength in a range from visible to infrared regions into the total reflection prism at an angle of incidence causing total reflection, and a measuring optical system receiving an outgoing beam from the total reflection prism for measuring absorbance by the sample mixed solution. The absorption in the total reflection is measured from intensity of attenuated total reflection.

The total reflection cell can be formed by a container type cell having only one opening for causing antigen-antibody reaction in the cell for forming a noble metal colloid labelled immune complex or making antigen-antibody reaction in another container for forming a noble metal colloid labelled immune complex and thereafter storing a sample mixed solution containing the noble metal colloid labelled immune complex, or a flow cell having a solution inlet port and a solution outlet port to be fed with a sample mixed solution after formation of a noble metal colloid labelled immune complex.

A light source included in the incident optical system can be formed by that emitting a beam of a continuous wavelength, or a single-wavelength light source such as a laser unit emitting a single-wavelength beam. When the light source emits a continuous wavelength beam, spectrometry can be carried out although the incident or measuring optical system must include a spectroscope. If a single-wavelength light source is employed, on the other hand, absorption measurement only at the single wavelength of the light source is carried out with no requirement for a spectroscope.

When only an antibody or an antigen which is labelled with a noble metal colloid is present in a sample mixed solution which is present in a total reflection cell, no absorption having sensitivity suitable for measurement is recognized. When an immune complex is not labelled with a noble metal colloid, no absorption having sensitivity suitable for measurement is recognized either. Absorption having sensitivity suitable for measurement is recognized at a specific wavelength only when an immune complex which is labelled with a noble metal colloid is present. The feature of the present invention resides in this point, whereby homogeneous immunoassay analysis is enabled.

Assuming that n₂ represents the refractive index of a sample mixed solution containing an immune complex and n₁ (n₂<n₁) represents the refractive index of a total reflection prism, a critical angle θc causing total reflection is expressed as follows: θc=sin⁻¹ (n ₂ /n ₁) When a measuring beam into the total reflection prism from an incident optical system is introduced, an angle θ of incidence (see FIG. 1) upon the interface between the total reflection prism and the sample mixed solution is set at the following condition: θ>θc

When homogeneous immunoassay analysis is carried out, a sample mixed solution which is subjected to absorption measurement is a mixed solution containing a noble metal colloid labelled antibody (or antigen), a noble metal colloid labelled immune complex, an unreacted antigen (or antibody), biopolymer and the like.

The measuring beam is that including a continuous wavelength beam in a proper region among near infrared, mid infrared and far infrared regions, or a single-wavelength beam.

Absorption in the total reflection prism is expressed by absorbance A as follows: A=N·α·de·loge where N represents the number of total reflection times in the total reflection prism, a represents the absorption coefficient of the sample mixed solution, and de represents an optical path length along which the measuring beam penetrates into the sample mixed solution in single total reflection.

According to the present invention, absorption by a noble metal colloid labelled immune complex which is present in a sample mixed solution is measured with a total reflection cell at high sensitivity. Absorption having sensitivity suitable for measurement is measured only in an immune complex which is in a state labelled with a noble metal colloid and no absorption having sensitivity suitable for measurement is recognized in only an antibody or an antigen which is labelled with a noble metal colloid or in an immune complex which is in a stable not labelled with a noble metal colloid, whereby qualification and determination of the immune material can be carried out by absorption by total reflection measurement without performing B-F separation, while immunoassay can be carried out through a simple operation and a simple measuring apparatus.

The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates an embodiment of the present invention;

FIG. 2 is a block diagram showing a measuring apparatus according to the embodiment;

FIG. 3 illustrates spectra of a gold colloid labelled immune complex and a gold colloid labelled antibody in the present invention;

FIG. 4 illustrates spectra of the gold colloid labelled immune complex in the present invention and an immune complex not labelled with a noble metal colloid;

FIG. 5 illustrates a total reflection absorption spectral difference spectrum of the gold colloid labelled immune complex in the present invention based on the spectra shown in FIG. 3;

FIG. 6 illustrates concentration dependency of absorbance at 2942.5 cm⁻¹ with respect to antibody IgG concentration in the immunoassay method according to the present invention;

FIG. 7 illustrates concentration dependency of absorbance at 2888.1 cm⁻¹ with respect to antibody IgG concentration in the immunoassay method according to the present invention;

FIG. 8 illustrates concentration dependency of absorbance at 1112.4 cm⁻¹ with respect to the antibody IgG concentration in the immunoassay method according to the present invention;

FIG. 9 illustrates concentration dependency of absorbance at 1043.3 cm⁻¹ with respect to the antibody IgG concentration in the immunoassay method according to the present invention;

FIG. 10 illustrates concentration dependency of absorbance at 993.8 cm⁻¹ with respect to antibody IgG concentration in the immunoassay method according to the present invention;

FIG. 11A is a front sectional view showing an exemplary total reflection cell having only one opening, and FIG. 11B shows a front sectional view and a plan view illustrating another total reflection cell having only one opening respectively; and

FIG. 12A is a perspective view and front sectional view showing illustrates an exemplary throwaway total reflection cell, and FIGS. 12B and 12E are perspective views and front sectional views showing other exemplary total reflection flow cells respectively.

FIG. 13 is a perspective view of a further embodiment of the total reflection cell.

FIG. 14 is a perspective view of another embodiment of the total reflection cell.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 schematically illustrates an embodiment of the present invention. A total reflection cell 2 has a total reflection prism 4 at least on one surface thereof. A mixed solution 8 containing a gold colloid labelled antibody 6 which is adsorbed on a gold colloid serving as an exemplary noble metal colloid is stored in the total reflection cell 2, and a sample solution 12 containing an antigen 10 causing antigen-antibody reaction with the antibody 6 is added thereto, so that the antibody 6 and the antigen 10 make antigen-antibody reaction in the mixed solution 8 stored in the total reflection cell 2, thereby forming a gold colloid labelled immune complex 14. A measuring beam is introduced into the total reflection prism 4 from an incident optical system 16 at an angle θ of incidence causing total reflection, whereby the measuring beam is transmitted through the total reflection prism 4 while being total reflected, and slightly penetrates into the mixed solution 8 through the interface between the total reflection prism 4 and the mixed solution 8, to be absorbed by the gold colloid labelled immune complex 14 in the mixed solution 8. An outgoing beam from the total reflection prism 4 is received by a measuring optical system 18 so that its absorbance is measured, whereby the antigen 10 contained in the sample solution 12 can be qualified and determined.

FIG. 2 schematically illustrates a measuring apparatus according to the embodiment. The incident optical system 16 includes a light source 16 a emitting the measuring beam and a beam adjusting optical system 16 b. The beam adjusting optical system 16 b includes an optical system for converting the beam from the light source 16 a to a parallel beam, a beam splitter for separating the beam into a measuring beam 20 s and reference beam 20 r, and an optical system for introducing the measuring beam 20 s into the total reflection prism 4 of the total reflection cell 2 at the angle of incidence causing total reflection. An optical system 22 for adjusting the luminous flux of the measuring beam 20 s which is totally reflected and transmitted through the total reflection prism 4 of the total reflection cell 2 and a spectroscope 23 for receiving the measuring beam 20 s adjusted by the optical system 22 and separating the received measuring beam into its spectral components are arranged on an optical path of the measuring beam 20 s, so that the measuring beam 20 s which is separated into its spectral components is guided to and detected by a detection part 26. The measuring optical system 18 shown in FIG. 1 includes the optical system 22, the spectroscope 23, and the detection part 26.

On the other hand, an optical system 24 for adjusting the luminous flux of the reference beam 20 r is arranged on an optical path of the reference beam 20 r for correcting fluctuation of the measuring beam 20 s, so that the adjusted reference beam 20 r is guided to and detected by the detection part 26. The detection part 26 is so formed as to correct the measuring beam 20 s which is transmitted through the total reflection prism 4 and separated into its spectral components through the spectroscope 23 by intensity of the reference beam 20 r indicating light source intensity thereby calculating absorbance.

Numeral 28 denotes a controller which controls the spectral operation of the spectroscope 23 for transmitting a detection output from the detection part 26 to a data processor 30. Numeral 32 denotes an output unit such as a recorder or a CRT (cathode-ray tube) outputting the result of processing in the data processor 30.

The light source 16 a can be formed by a fluorescent lamp, a xenon lamp, a black-body radiation source, a gas laser, a solid laser or a semiconductor laser.

The material for the total reflection prism 4 can be prepared from ZnSe, Ge, Si, Al₂O₃ or MgO. Only the total reflection prism 4 or overall wall surfaces of the total reflection cell 22 including the total reflection prism 4 may be made of such a material.

The spectroscope 23 can be formed by an FTIR (Fourier transformation infrared spectrophotometer), a system which is employing IR-blazed grating and IR-sensitive detector or the like. The spectroscope 23 may be included in the incident optical system 16. When a single-wavelength light source emitting a beam of a single wavelength is employed as the light source 16 a, the spectroscope 23 and the controller 28 for controlling the same are unnecessary.

The cell 2 shown in FIG. 1 was employed, the spectroscope 23 of the measuring apparatus shown in FIG. 2 was formed by an FTIR, a ZnSe optical crystal was employed as the total reflection prism 4 of the cell 2, Anti-Mouse IgG (product by BioCell (U.S.A.)) absorbed on a gold colloid of 30 nm in grain size was employed as the noble metal colloid labelled antibody 6, and a mixed solution (pH 7.65) of 0.01 M of TWEEN 21 and PBS (phosphate-buffered saline) was employed as a solvent for a mixed solution containing the antibody 6. Detection was carried out 10 times in a single measuring time of 2 minutes, and the results of the detection were estimated. The spectroscope 23 had a spectral range of 800 to 3200 cm⁻¹ and resolution of 2 cm⁻¹.

FIGS. 3 and 4 shows total reflection spectra of the results of the measurement. Referring to FIG. 3, a spectrum {circle around (2)} is that of a mixed solution containing only the aforementioned 30 nm gold colloid labelled Anti-Mouse IgG. Large absorption is that of water, while no another characteristic absorption is recognized. Referring to FIG. 4, on the other hand, a spectrum {circle around (3)} is that of a mixed solution of 2 mg/ml in concentration containing only an IgG immune complex not labelled with a noble metal colloid. No characteristic absorption is recognized in the spectrum {circle around (3)} either, except that of water. On the other hand, a spectrum {circle around (1)} appearing in each of FIGS. 3 and 4 is that of a mixed solution containing a gold colloid labelled immune complex formed by reaction of 7.5 μg/ml of the aforementioned 30 nm gold colloid labelled Anti-Mouse IgG antibody and 62.5 μg/ml of a Mouse IgG antigen, and characteristic absorption is recognized in addition to that of water.

FIG. 5 illustrates a spectrum obtained by subtracting the spectrum {circle around (2)} from the spectrum {circle around (1)} in FIG. 3 as a background, in order to clearly show the absorption. This spectrum is a total reflection absorption spectral difference spectrum of the gold colloid labelled immune complex formed by reaction of 7.5 μg/ml of the 30 nm gold colloid labelled Anti-Mouse IgG antibody and 62.5 μg/ml of the Mouse IgG antigen. In this spectrum, characteristic absorption bands appear at 2888 cm⁻¹ and 2942 cm⁻¹ in the vicinity of 2900 cm⁻¹, and 993 cm⁻¹, 1043 cm⁻¹ and 1112 cm⁻¹ between the vicinity of 900 cm⁻¹ and 1500 cm⁻¹, and this is absorption by internal vibration of the IgG immune complex.

FIGS. 6 to 10 illustrate concentration dependency levels of absorbance values at respective wavenumbers of 2942.5 cm⁻¹, 2881.1 cm⁻¹, 1112.4 cm⁻¹, 1043.3 cm⁻¹ and 993.8 cm⁻¹ as to various concentration values of Mouse IgG antigen reacted with 7.5 μg/ml of 30 nm gold colloid labelled Anti-Mouse IgG antibodies respectively. The axes of abscissas show IgG concentration values, and the axes of ordinates show absorbance values. Every one is saturated in a high concentration region of IgG, while there is a linear relation in a low concentration region, and this shows that quantitative analysis is possible by the inventive method.

FIGS. 11A and 11B show exemplary total reflection cells having only single openings. FIG. 11A illustrates the cell 2 shown in FIG. 1, having the total reflection prism 4 on its bottom surface.

FIG. 11B shows a front sectional view and a top plan view of a cell 40 having a total reflection prism 4 on its side surface.

FIGS. 12A to 12E show perspective views and front sectional views of cells having other shapes respectively.

FIG. 12A shows a single-sided throwaway cell, which is provided with a narrow clearance 42 for sucking a sample mixed solution by a capillary phenomenon and a total reflection prism 4 formed along the clearance 42. Numeral 15 denotes the sample mixed solution which is sucked in the clearance 42.

FIG. 12B shows an exemplary double-sided total reflection flow cell, which is provided on upper and lower surfaces thereof with total reflection prisms 4 and 4′ through a space fed with a sample mixed solution. Numeral 44 denotes a sample mixed solution inlet port for the cell, and numeral 46 denotes a sample mixed solution outlet port from the cell.

FIG. 12C shows an exemplary cylindrical total reflection flow cell, which is formed to enclose a side surface of a cylindrical total reflection prism 4. A sample mixed solution 15 is fed along the cylindrical surface of the total reflection prism 4.

FIG. 12D shows another exemplary double-sided total reflection flow cell, which is so formed that a sample mixed solution 15 is fed along two opposite surfaces of a total reflection prism 4 respectively.

FIG. 12E shows an exemplary single-sided total reflection flow cell, having a total reflection prism 4 on one surface defining a space fed with a sample mixed solution 15.

FIG. 13 shows a further embodiment of the total reflection cell which is provided with a narrow clearance 42 for introducing a sample solution thereinto. Total reflection prism 4 a has an incident plane 50 and a reflecting mirror plane 52 on an opposite side to incident plane 50. The measuring beam introduced into prism 4 a from incident 50 is transmitted while totally reflecting through prism 4 a to mirror lane 52, and reflected by mirror plane 52 to be transmitted back to incident plane 50 while totally reflecting, and finally outdoes from incident plane 50.

FIG. 14 shows a still further embodiment of the total reflection cell which is provided with a narrow clearance 42, as described above. Total reflection prism 4 b has an incident plane 54 provided with reflecting mirror coating except for a part 56 of incidence of a measuring beam and an outgoing plane 58 provided with reflecting mirror coating except for a part 60 of outgoing of the measuring beam. Outgoing part 60 is apart from the opposite position to incidence part 56. The measuring beam introduced into prism 4 b from incident part 56 is transmitted repeatedly while total reflecting between mirror planes 54 and 58, and finally outgoes from outgoing part 60.

According to the embodiments of FIGS. 13 and 14, the number of total reflection is increased to improve the sensitivity of measuring absorption.

A still further embodiment is a throwaway (i.e. disposable) cell prepared from plastics and provided with a noble metal colloidal on its inner surface. When a sample solution is introduced into this cell, the immune complex in the sample solution is adsorbed on the noble metal colloidal of the cell, and absorption of the immune complex is measured in a labeled state by the metal colloidal.

According to the embodiment of this throwaway cell, a sample solution containing immune complex, which is not labeled by noble metal colloidal, can be measured directly.

Although the present invention has been described and illustrated in detail, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, the spirit and scope of the present invention being limited only by the terms of the appended claims. 

1. An immunoassay apparatus comprising: a total reflection cell having at least one total reflection prism consisting of a material having a large refractive index than a sample mixed solution containing an immune complex being labelled with a noble metal colloid, wherein said at least one total reflection prism is on at least one wall surface which defines a space in which said sample mixed solution is present; an incident optical system configured to introduce a measuring beam of a wavelength in a range from visible to infrared regions into said at least one total reflection prism at an angle of incidence causing total reflection; and a measuring optical system configured to receive an outgoing beam from said at least one total reflection prism for measuring absorbance by said sample mixed solution.
 2. The immunoassay apparatus in accordance with claim 1, wherein said total reflection cell has only one opening.
 3. The immunoassay apparatus in accordance with claim 1, wherein said total reflection cell is a flow cell having a solution inlet port and a solution outlet port, being fed with said sample mixed solution after formation of said immune complex being labelled with said noble metal colloid.
 4. The immunoassay apparatus in accordance with claim 1, wherein said incident optical system includes a light source configured to emit a beam of a continuous wavelength, and said incident or measuring optical system includes a spectroscope for carrying out spectrometry.
 5. The immunoassay apparatus in accordance with claim 1, wherein said incident optical system includes a single-wavelength light source configured to emit a single-wavelength beam, and said incident and measuring optical systems include no spectroscopes, so that absorption measurement is carried out only at said single wavelength of said light source.
 6. The immunoassay apparatus in accordance with claim 1, wherein said total reflection cell is a double-sided total refection flow cell having total reflection prisms on upper and lower wall surfaces thereof.
 7. The immunoassay apparatus in accordance with claim 1, wherein said total reflection cell is a cylindrical total reflection flow cell having a cylindrical body and a cylindrical total reflection prism enclosed therein, wherein an inner surface of said cylindrical body and an outer surface of said total reflection prism together define an annular flow space in which said sample mixed solution is present.
 8. The immunoassay apparatus in accordance with claim 1, wherein said total reflection cell is a double-sided total reflection flow cell having total reflection prisms enclosed therein, wherein inner wall surfaces of said total reflection flow cell and outer surfaces of said total reflection prisms together define flow spaces on opposite sides of said total reflection prisms in which said sample mixed solution is present.
 9. The immunoassay apparatus in accordance with claim 1, wherein said at least one total reflective prism has an incident plane at a first planar end thereof and comprises a reflecting mirror at a second planar end thereof configured to reflect said measuring beam toward said incident plane; and said incident optical system is configured to introduce said measuring beam into said incident plane.
 10. The immunoassay apparatus in accordance with claim 1, wherein said at least one total reflective prism has (i) an inlet port into which said measuring beam is introduced and (ii) a first reflecting mirror at an incident plane thereof and (i) an outlet port and (ii) a second reflecting mirror at an outgoing plane thereof, wherein said second reflecting mirror is configured to reflect said measuring beam toward said incident plane and said first reflecting mirror is configured to reflect said measuring beam toward said outgoing plane; and said measuring optical system is configured to receive said outgoing beam from said outlet port.
 11. The immunoassay apparatus in accordance with claim 1, wherein said total reflection cell is a throwaway cell provided with a noble metal colloid on said at least one wall surface. 